Static Pressure-Volume Curves for the Lung of the Frog (Rana Pipiens)

During the past 30 years evidence has accumulated supporting the view that the lungs of mammals are lined with a layer of lipids and proteins, probably as lipoprotein, whose main function is to reduce the surface tension of the curved alveolar surfaces. The first indication of such a lining was probably that obtained by von Neergaard in 1929 when he demonstrated that the pressure necessary to enlarge the lungs to a given volume was less when they were filled with liquid than when they were inflated with air. Replacement of the air-alveolar interface by a liquid-alveolar interface evidently produced a significant reduction in the retractive forces operating on the lung, von Neergaard concluded that surface tension forces were important in the lungs. From pressure/volume experiments approximate calculations were made which suggested that a considerable surface tension (about 40–50 dyn/cm^†) is present at such air-alveolar interfaces (Radford, 1954; Brown, 1957). Brown (1957) who knew about the lining film, calculated a much lower surface tension in deflated lungs. Since these initial experiments this hypothesis has been supported by several other lines of evidence including electron-microscopic studies of the lung lining layers, chemical analyses of lung tissue and of washings from the lungs and also from direct measurements of the surface tension using a Wilhelmy balance (Clements, 1957), or a bubble technique (Pattle, 1958). Corresponding studies with the lungs of lower vertebrates are as yet fragmentary, but in general support the view that similar lining layers are present in lungfish, amphibians, reptiles and birds (Brooks, 1970; Clements, 1962; Clements, Nellengoben & Trahan, 1970; Hallman & Gluck, 1976; Hughes, 1967, 1970, 1973; Pattle, Schock, Creasey & Hughes 1977). However, there have been few studies (Cragg, 1975) of pressure-volume relationships corresponding to the initial work of von Neergaard. The purpose of the present investigations was to determine these relationships for lungs filled with air and saline and as a result to make estimates of the surface tension of the lung lining. A preliminary report of this work has been given (Vergara & Hughes, 1977).

Experiments were carried out on frogs (Rana pipiens)of 15–35 g body weight. The animals were maintained in the laboratory at 13–14 °C and before cannulation of the lung they were anaesthetized by an intra-peritoneal injection of a 0·1 ml saline solution containing 10% MS 222. A cannula (PP120) was flared at one end and inserted into the glottis where it was tied firmly. In some experiments inflation and deflation of the lung was carried out using the intact frog. In most cases, however, the frog was dissected carefully from the ventral side and one of the lungs isolated together with its connexion to the cannula. With most preparations, it was only possible effectively to cannulate a single lung in any given specimen. The isolated lung was connected into the apparatus shown in Figs. 1 and 2 and usually was used for air inflation and deflation experiments before being filled with saline. The apparatus and procedures were similar to those used by Young, Tierney & Clements (1970) and by Cragg (1975). Results obtained in the last of the air P/V curves were compared with those obtained for the second of the saline curves in order to calculate surface tension. Many lungs contained parasites and in no case were such lungs used for the experiments.

Inflation with saline (0 ·9 % NaCl) was carried out using the apparatus in Fig. 2. First of all the pressure in the isolated lung was reduced to – 20 cm H₂O using the air apparatus (Fig. 1). It was then attached to the saline apparatus at this reduced pressure with the lung at a minimal volume. After adjusting (syringe P) the pressure within the saline apparatus to the same level (– 20 cm H₂O) connexion was made to the lung. Inflation of the lung with saline (syringe I/D) was once more carried out at steps of 0·1–0·2 ml, 3 min being taken for each step. As saline was injected into the lung the pressure was read off on MI, as it approached atmospheric pressure and the condition indicated in Fig. 2. Inflation above atmospheric pressure was continued and followed by deflation. During the saline inflation/deflation experiments the lung remained submerged in saline.

Before the apparatus was used for experiments with frog lungs some control studies were made to determine the pressure/volume curves of rubber balloons using air and saline (Fig. 3). The results indicated that there was no hysteresis in the pressure/ volume relationship because during both inflation and deflation with either air or saline, the sigmoid-shaped curves were perfectly matched. Similar curves could be obtained provided the balloons were used on no more than three or four occasions.

Lungs were fixed at different inflation volumes by immersion in a 2·5 % glutaraldehyde solution in a cacodylate buffer (pH 7·2) before embedding in an Epon-Araldite mixture. Large sections, covering the whole cross-section, were cut with a LKB ultratome and stained with toludine blue. These sections were analysed morphometrically (Hughes & Weibel, 1976) by means of intersection and point counting using a projection microscope.

During inflation of the lung from atmospheric pressure, the increment in pressure per unit volume change decreased rapidly until there was very little change in pressure although lung volume increased very significantly. Above this level the curve showed its sigmoid nature as the pressure rose more steeply during the final 1 ml of inflation. The deflation curve was similar in general shape but was shifted to the left relative to the inflation curve (Fig. 4). Consequently the pressure at any given volume is less during deflation than during inflation. This marked hysteresis was found in all preparations. Differences in the precise nature of the curves were observed when repeated inflation and deflation cycles were carried out.

The general form of the curves is similar to that obtained with air but during both inflation and deflation the pressure changes were very much less for any given volume change. The sigmoid shape of these curves was less pronounced with saline-filled lungs as also was the hysteresis (Fig. 4). Although variations were found in the precise nature of these curves according to the size of the frog, the same qualitative differences were always observed. There was no significant change in the shape or the hysteresis of the P/V curve when the rate of change of volume was altered. In Fig. 5 the inflation of the curves are plotted and a line drawn to indicate the difference in pressure at one particular volume.

Pressure/volume curves showing hysteresis for lungs in the whole animal are shown in Fig. 6 for comparison with those obtained for isolated preparations. The curves obtained from the intact animal (Fig. 6 a) did not change very markedly when the frog was opened up and the air pressure/volume relationship once more determined (Fig. 6 b). But when the heart, pericardium and liver were removed the resting volume of the lung at atmospheric pressure was reduced (Fig. 6c). Thus in the whole animal a greater pressure was required for the same volume change-presumably because of the limitations imposed by the viscera and body wall.

From the pressure/volume curve described above it is possible to obtain values for lung compliance. It is evident that the compliance during saline filling is greater than during filling with air, the pressure changes for a given volume of saline injection being much less than those following a comparable volume change produced by air injection. The compliance and area of the hysteresis loop during air filling were almost identical both before and after the lung had been inflated with saline.

Estimates of compliance usually indicate the slope of some part of the pressure/ volume curve. In the frog little data is available concerning the range of volume changes during normal ventilation. Calculations were therefore made on all parts of the pressure/volume curves for the isolated and in situ lungs. Fig. 7 shows plots of the compliance in relation to the relative volume ; each point represents the compliance at intervals of 1 cm H₂O pressure change. The maximum compliance was found when the volume of the lung was about half its maximum inflation, i.e. 50% of the volume at which an increase in pressure gave scarcely any increase in volume. The minimum values occurred at the end of inflation and the beginning of deflation (i.e. 100% relative volume). During deflation the value rose to approximately the same maximum as during inflation. An approximately tenfold range of compliance was found during inflation for any frog lung. Analysis of the pressure/volume curves from intact preparations (Fig. 8) gave essentially the same results, but the whole range of values was slightly lower. It is evident from the plots given in Fig. 7 that values for the frog are about ten times greater than corresponding values for the mouse or lizard of approximately the same body mass.

As a result of the investigations on the frog lung an interest developed in the range of compliance values obtained in different parts of the pressure/volume curve, and a comparison with results obtained for mammals. Values normally given for the mammal are in relation to the functional residual capacity (shown as the first point plotted in Fig. 7), but for comparative purposes a number of published pressure/ volume curves have been analysed. Maximum and minimum values for compliance derived from such curves are summarized in Table 1. In making comparative studies scaling factors must always be taken into account and for mammals (Stahl, 1967) the relationship has been shown to be :

During the present survey a similar relationship has been shown for maximum compliance (= 0·00244 W^1·049) determined from the P/V curves of mammals and a relationship having a similar slope (= 0·036 W^0·994) was obtained for lower vertebrates (Fig. 9). The tenfold difference in the intercept values for these regression lines indicates that at almost all body sizes a lower vertebrate lung would be expected to have a compliance that is approximately ten times that for a mammal of comparable body size. Thus the deduction based upon the relationships between frog and mouse lungs (Fig. 7) is given further support.

It is also possible to compare the result obtained from these static pressure/volume relationships with the P/V curves obtained by West & Jones (1975) for the dynamic relationships recorded during pulmonary ventilation. Calculations of compliance for different parts of their curves suggest a similar range of values (Fig. 8). Thus it can be concluded that the values of compliance obtained in this study give a good indication of the type of compliance relationship which operates in the normal pulmonary ventilation of Rana pipiens. Presumably the greater compliance of the frog lung reduces the work of breathing.

The general form of the pressure/volume curves obtained using both air and saline was very similar and showed a hysteresis in all cases. The pressure required for inflation to a given volume using saline was always less than with air. These observations support the view that the lung lining contains a substance which reduces surface tension forces within the lung. The finding that no such differences in pressure/volume curves were observed in balloons inflated with air or saline is also in agreement with this interpretation.

Assuming that the forces due to tissue elasticity remain the same when the lung is filled with air or saline and that there are no changes in the basic geometry of the alveoli and other air spaces, then it is probable that the only mechanisms responsible for the difference in pressure/volume relationships are due to the surface forces at the air-liquid interface.

From each pair of air and saline pressure/volume curves calculations were made of the surface tension using a method based upon that of Fisher, Wilson & Weber (1970) and Bachofen, Hildebrandt & Bachofen (1970). In this method it is necessary to assume a maximum value for the surface tension, and for this purpose the value used (50 mN/m) was based upon measurements of the surface tension of washings from mammalian lung using a Wilhelmy surface tension balance (Table 2). It has often been assumed that the internal surface area of a vertebrate lung (mammal) is proportional to the two-thirds power of the lung volume. In a lung containing large numbers of alveoli approximately spherical in shape, such an assumption is reasonable, but for a lung so different in basic organization as that of the frog it was decided to investigate this relationship in more detail. Morphometric estimates of the air volume were obtained from point counts of sectioned material and an estimate of the internal surface area was obtained from intersection counting. Results obtained for lungs of different inflation volumes showed that the surface area is more closely related to the one-half power of the volume (SA ∝V^0·52).

It is evident from the calculations summarized above that insertion of values for K and appropriate volumes and P_s in equation (3) gives the surface tension at different volumes. Some of the values obtained are given in Table 2, together with the results of surface tension measurements on a variety of tetrapod lungs using different methods.

Changes in surface tension associated with changing surface area plotted out as a change in relative area, calculated on the basis of the power relationship to lung volume are given in Fig. 10. This plot shows a hysteresis loop which encloses a smaller area than that obtained for a mammalian lung (Fisher et al. 1970).

When interpreting the curves relating surface tension to surface area for both the frog and mammalian lungs it is apparent that the fall in surface tension is very rapid following a decrease of only 15–20 % in surface area. This contrasts with the results obtained with the surface-tension balance, using washings or extracts of mammalian lungs, where a much greater reduction in area, by about 40–50 % of its original area, is necessary to obtain a comparable reduction in surface tension, i.e. to about 5–10 mN / m. When the lung volume, and consequently surface area, is reduced to about 50% of the total lung capacity, the surface tension falls to about 5 % of its maximum value. At small volumes, however, the differences in pressure between the air and saline deflation curves approach zero and consequently the surface tension must also approach zero. During re-expansion of the lung the surface tension rises once more but at any given surface area is always less during deflation than during inflation. Miller & Bondurant (1961) studied the surface tension/area loop of extracts of frog lung with a Wilhelmy balance. They found a minimum value of 30 dyn/cm when the surface was reduced to 20 % of its original area. The area of the hysteresis loop was half that found for rat lungs under comparable conditions. No mention was made of the temperature at which these experiments were carried out. This parameter has a very important effect on surface activity and has been shown to be especially relevant in relation to the properties of frog lung surfactant (Pattle et al. 1977). The experiments described in the present paper were carried out at 13 °C. However, it is difficult to compare satisfactorily the loops obtained with a surface balance by Miller and Bondurant and those described here using P/V curves, as many authors have found a discrepancy between the results of the two methods for mammalian lungs.

One advantage of the present study is that it is based on a morphometric determination of the relationship between surface area and volume of the lung without any assumption concerned with the shape of the basic units.

In conclusion it may be stated that evidence for the existence of a surfactant lining in the frog lung has now been increased by the results of these pressure/volume studies. Added to the known evidence derived from electron microscopy, and measurements on bubbles squeezed from frog lungs, it seems safe to conclude that such a lining is present. The possible function of such a lining is not very clear at first sight. The function usually applicable to the mammalian lung does not seem ideal because of the much larger dimensions of the alveolar units. Nevertheless, it is very probable that the presence of a surface tension reducing film would play an important role in the early stage of inflation of these lungs, especially in small frogs, for they are almost completely emptied during expiration.

It was noticeable during the inflation experiments that the lung tended to fill first in its more anterior regions, i.e. those regions closest to the glottis, and perhaps during such a stage of the ventilatory cycle the lining film would be important.

The range of the pressure and volume changes employed in the present study are clearly in excess of those of the normal ventilatory cycle. However, it is well known that the lungs of frogs are inflated to volumes which greatly exceed the normal dimensions at full inspiration in certain stages of the life-cycle, e.g. vocalization during the breeding season. The lungs are also inflated supramaximally to serve a buoyancy function. Thus it can be concluded that the total range of pressure/volume relationships shown here approximates to that which normally occurs in such structures.

From comparison of washings obtained from frog lungs with washings from mammalian lungs (Hughes & Vergara, 1978), it appears that the lining layers are more similar in their composition than was at first supposed and this becomes especially apparent when the concentrations of the different phospholipid components are compared in relation to the area of their internal surfaces. Thus, in spite of the differences in alveolar dimensions, these similarities suggest that perhaps the lining layers may perform similar functions. Consequently the supposed role of such layers in reducing the surface tension forces may have been over-emphasized. An ‘antiglue’ function of a surfactant lining is clearly a possibility, as has been suggested for the mammalian lung (Sanderson et al. 1976). In the case of the mammalian lung, some morphometric and physiological investigations (Weibel et al. 1973; Hills, 1971) have suggested that many alveoli collapse at different stages during the ventilatory cycle and the alveolar linings slide over one another as they open and close. Similar geometric modifications might also take place in lungs of the frog type (Hughes, 1978); in fact material fixed at different levels of inflation has confirmed this possibility. Until further evidence is forthcoming such changes in overall lung morphology must be speculative, but at least such an hypothesis has the advantage of enabling us to understand similarities between lungs which may have wide differences in their surface properties because of their dimensions alone.

We wish to thank the Medical Research Council for providing a research grant which made it possible to carry out this work.

We are grateful to Professor Robert Bils for skilfully cutting the large sections of frog lungs.